EP1142902A2 - Analogues des oligonucléotides dérivées en position 3', avec des groupes non-nucléotidiques, leur préparation et leur utilisation - Google Patents

Analogues des oligonucléotides dérivées en position 3', avec des groupes non-nucléotidiques, leur préparation et leur utilisation Download PDF

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EP1142902A2
EP1142902A2 EP01116471A EP01116471A EP1142902A2 EP 1142902 A2 EP1142902 A2 EP 1142902A2 EP 01116471 A EP01116471 A EP 01116471A EP 01116471 A EP01116471 A EP 01116471A EP 1142902 A2 EP1142902 A2 EP 1142902A2
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alkyl
formula
aryl
integer
hydroxy
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EP1142902B1 (fr
EP1142902A3 (fr
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Anuschirwan Dr. Peyman
Eugen Dr. Uhlmann
Irvin Dr. Winkler
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Sanofi Aventis Deutschland GmbH
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Hoechst AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel oligonucleotide analogs with valuable physical, biological and pharmacological properties as well as a Process for their production.
  • Your application relates to use as Inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense Oligonucleotides and Triplex Forming Oligonucleotides), as probes for the detection of Nucleic acids and as an aid in molecular biology.
  • Oligonucleotides are increasingly used as inhibitors of Gene Expression (G. Zon, Pharmaceutical Research 5, 539 (1988); J. S. Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press; C. Helene and J. J. Toulme, Biochemica et Biophysica Acta 1049, 99 (1990); E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990)).
  • antisense oligonucleotides Nucleic acid fragments whose base sequence is complementary to one inhibiting mRNA. This target mRNA can be cellular, viral or other be of pathogenic origin.
  • Examples of cellular target sequences are: those of receptors, enzymes, immunomodulators, ion channels or oncogenes in Question.
  • Inhibition of Virus Propagation Using Antisense Oligonucleotides was for example for RSV (Rous Sarcoma Virus), HSV-1 and -2 (Herpes Simplex Virus types I and II), HIV (Human Immunodeficiency Virus) and influenza viruses described. In doing so, one uses oligonucleotides that form the viral nucleic acid are complementary.
  • Sense oligonucleotides are so in their sequence designed to be, for example, nucleic acid binding proteins or Nucleic acid-processing enzymes bind ("capture") and thus their biological Inhibit activity (Helene, 1990).
  • the viral targets here are, for example To name reverse transcriptase, DNA polymerase and transactivator proteins.
  • Triplex forming oligonucleotides generally target and form DNA a triple helical structure after binding to it.
  • the antisense and Triplex binding regions can either be in two separate oligonucleotides or be housed in an oligonucleotide.
  • Another application Synthetic oligonucleotides are the so-called ribozymes, which are the target RNA due to their ribonuclease activity (J.J. Rossi and N. Sarver, TIBTECH 8, 179 (1990).
  • nucleic acid fragments are labeled as suitable so-called DNA probes or DNA probes for the specific hybridization to a Detected nucleic acid used.
  • the specific training of the new The double strand is made with the aid of the label, which is preferably not radioactive is persecuted. This way, genetic, malignant, viral or other Detect pathogenic diseases.
  • oligonucleotides usually takes place in such a way that Phosphate backbone, ribose unit or the nucleobases changed accordingly (Cohen, 1989; Uhlmann and Peyman, 1990).
  • Another commonly used Method is the preparation of oligonucleotide-5 'conjugates by reaction of the 5'-hydroxy group with appropriate phosphorylation reagents.
  • Oligonucleotides that are only modified at the 5 'end have the disadvantage that they are in the Serum are broken down. If, however, all internucleotide phosphate residues are changed the properties of the oligonucleotides often change drastically.
  • the solubility of the methylphosphonate oligonucleotides in aqueous Medium is reduced and the hybridization capacity is reduced.
  • Phosphorothioate oligonucleotides act nonspecifically, so that for example Homooligomers are effective against viruses.
  • Antisense oligonucleotides generally have a uniform polarity, which usually has an antiparallel character when hybridized to RNA (see Table 1; A). In certain cases, for example in the case of oligonucleotides composed of a-nucleoside units, the polarity can also be parallel (Table 1; B). Triplex forming oligonucleotides can generally hybridize to double-stranded nucleic acids in a sequence-dependent manner in parallel (Table 1; C) or antiparallel (Table 1; D) orientation with respect to the purine-rich nucleic acid strand.
  • the base pairing motifs T • AT, G • GC, C + • GC, G • TA, C Me • GC, A • AT and C PI • GC are used, in which C + is a protonated cytosine residue, C Me is a 5 -Methyl cytosine residue, C PI a pseudoisocytosine residue and "•" a Hoogsteen or reverse Hoogsteen base pairing.
  • the use of these Hoogsteen base pairs is limited to purine-rich regions of the double-stranded nucleic acids.
  • oligonucleotides with alternating polarity were produced which are due to a (5'5 ') strand change (Table 1; E) or a (3'3') - strand change can alternate if necessary (Table 1; F), can bind purine-rich regions of the opposite strand (Ono et al., Biochemistry (1991) 30, 9914).
  • a strand change while maintaining the polarity is possible if a (3'5 ') spacer is installed.
  • oligonucleotides can be produced which contain a 5'5 'spacer (Table 1; K) at the 5' end.
  • oligonucleotide analogs both 3 '(2') ends can advantageously contain phosphoryl groups.
  • oligonucleotide analogs which the in Table 1 C to I Obey the principles of action shown, generally have one Hydroxy group at the 3 'end, so that they are broken down in the serum, mostly are poorly membrane-permeable and can only be easily derivatized at the 5 'end.
  • the task is therefore to use specific oligonucleotide analogs Hybridization properties against single and double stranded nucleic acids, increased serum stability, good solubility and specific effectiveness to provide.
  • Preferred oligonucleotide analogs of the formula IB are those in which the base B is in the ⁇ position, the nucleotides are in the D configuration, R 2 is in the 2 'position and a is oxy.
  • oligonucleotide analogs of the formula IB in which V, Y 'and Y have the meaning of oxy.
  • Oligonucleotide analogs of the formula IB are furthermore particularly preferred, where V, Y, Y 'and W have the meaning of oxy and oxo, respectively.
  • Oligonucleotide analogs of the formula IB in which R 1 is hydrogen are also preferred.
  • oligonucleotide analogs of the formula IB in which U, V, W, X, Y 'and Y have the meaning of oxy, oxo or hydroxy and R 1 is hydrogen.
  • the recurring residues such as R 2 , B, a, d, e, f, g, g ', h, W, V, Y, Y', U, R 3 , R 4 , p, q, G and Z can independently of one another have identical or different meanings, ie, for example, V independently of one another means oxy, sulfanediyl or imino.
  • Halogen is preferably fluorine, chlorine or bromine.
  • Heteroaryl is understood to mean the remainder of a monocyclic or bicyclic (C 3 -C 9 ) heteroaromatic which contains one or two N atoms and / or an S or an O atom in the ring system.
  • Typical marker groups are:
  • Oligonucleotide analogs which bind or intercalate and / or cleave or crosslink to nucleic acids contain e.g. B. acridine, psoralen, phenanthridine, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.
  • Typical intercalating and cross-linking residues are:
  • Examples of groups NR 3 R 4 in which R 3 and R 4 together with the nitrogen atom carrying them form a 5- to 6-membered heterocyclic ring which additionally contains a further heteroatom are the morpholinyl and the imidazolidinyl radical .
  • the invention is not based on ⁇ - and ⁇ -D- or L-ribofuranosides, ⁇ - and ⁇ -D- or L-deoxyribofuranosides and corresponding five-ring carbocyclic analogues limited, but also applies to oligonucleotide analogs that come from others Sugar building blocks are built, for example ring-expanded and ring-narrowed Sugar, acyclic or other suitable sugar derivatives.
  • the invention is furthermore not to the derivatives of Limited phosphate residue, but also refers to the known Dephospho derivatives.
  • Oligonucleotide analogs of Formula IB have a 3'5 'spacer (3'5'S) or (2'5'S) that does not have the polarity influenced, but for example a refolding of the oligonucleotide analog or a strand change allowed.
  • S propane-1,3-diol phosphate, 2-benzyl and 2-octadecyl oxypropane 1.3-diolphosphate, triethylene glycol or hexaethylene glycol phosphates, which are can also repeat if necessary.
  • Nucleotide analogs without heterocyclic Base and phenylene dialkylene residues are further preferred embodiments of S, especially from (3'3'S).
  • (5'5'S) is for topological reasons longer than (3'3'S). So (5'5'S) usually has 20 to 45, preferably 24 to 36 unbranched bonds, while (3'3'S) only 5 to 15, preferably 6 to 10 has unbranched bonds, provided that a strand change is sought.
  • S serves to refold the oligonucleotide analog, for example for triplex formation on a single strand of nucleic acid
  • S are lengths of S. correspondingly from 2 to 8, preferably from 4 to 5 nucleotide units is advantageous.
  • examples include penta- (2-benzyloxy-1,3-propanediol) hexaphosphate and Hexa- (propane-1,3-diol) pentaphosphate mentioned, each one (5'5'S) - or (3'5'S) linkage can cause.
  • oligonucleotide analogs of the formula B is carried out similarly to the synthesis of biological oligonucleotides in solution or preferably in solid Phase, if necessary with the help of an automatic synthesizer.
  • Solid phase synthesis of oligonucleotides with a phosphate or Phosphate ester residue at the 3 'end is according to the standard phosphoramidite chemistry according to Caruthers (M.D. Matteucci and M.H. Caruthers, J. Am. Chem. Soc. 103, 3185 (1981)) not possible because the first nucleotide building block is via the 3'-hydroxy group is bound to the solid support and therefore from this Syntheses always result in oligonucleotides with a 3'-hydroxy group.
  • It different methods according to the solid phase method have been described but all are cumbersome and often do not have derivatives such as phosphate esters or have alkylphosphonates prepared (R.
  • a solid support of the formula IV is used as the starting component for the solid phase synthesis D-X'-CH 2 CH 2 -S (O) x -CH 2 CH 2 -AT wherein A stands for a linker arm which, for example, is a radical of a dicarboxylic acid, a diol, an alkylamine, a dicarboxylic acid monoalkylamide, an acid amide or a phosphate of the formula
  • R hydrogen or C 1 -C 6 alkyl, which is optionally substituted by -CN, preferably methyl or 2-cyanoethyl
  • T is a solid support, for example made of materials such as CPG (Controlled Pore Glass), silica gel or an organic resin such as polystyrene (PS) or a graft copolymer of PS and polyethylene glycol (POE), which is modified by functional groups such as hydroxy, amino, halogen or COOH in the side chain
  • D stands for a protective group which can be removed without cleaving the linker arm A and the X'-CH 2 CH 2 -S (O) x -CH 2 CH 2 residue (see Bioorg. Chem.
  • the linker arm A which connects the solid support T with the sulfur-containing residue by chemical bonding (amide, ester, etc.) (Damka et al., Nucleic Acids Res. 18, 3813 (1990)), is preferably a succinic acid residue (OC (O) -CH 2 CH 2 -C (O) -), an oxalic acid residue, (OC (O) -C (O) -), an alkylamine, preferably LCAA (long chain alkyl amine), or polyethylene glycol.
  • a succinic acid residue is particularly preferred. In certain cases, for example in combination with substituents that cannot withstand prolonged ammonia treatment, more unstable linkers such as the oxalyl linker are advantageous.
  • the preparation of solid supports of the formulas IV ac is described in Example 1.
  • Preferred bis-amidites are those of the formula Vla
  • the introduction of the group Z can also be carried out according to the H-phosphonate method, in that the first by reaction of a nucleoside phosphonate of the formula Xl wherein R, V, a, B ', Y', X 'and W have the meaning given above, H-phosphonate diesters of the formula VII' formed with a support of the formula IV ' is subjected to an oxidative phosphoramidation (B. Froehler, Tetrahedron Lett. 27, 5575 (1986). In this way, for example, an oligonucleotide with a 3'-terminal cholesteryl group can be produced with cholesteryl-oxycarbonyl-aminoalkylamine in the presence of carbon tetrachloride.
  • oligonucleotide analogs of the formulas IB is also possible according to the Triester method, in which the group HX 'of the support of the formula IV' is protected with a protected phosphate diester of the formula XII wherein R, V, a, B ', R 2 , Y', Z, W, X 'and the curly bracket have the meaning given above, in the presence of a condensing agent such as arylsulfonyl chloride and a nucleophilic catalyst such as tetrazole.
  • a condensing agent such as arylsulfonyl chloride
  • a nucleophilic catalyst such as tetrazole.
  • oligonucleotide chain can, if necessary, be successively coupled with building blocks of the formula XIII wherein U 'has the meaning of U, with the proviso that U' is not NHR 3 or NR 3 R 4 and hydroxy, mercapto and SeH are protected derivatives (for examples of such derivatives see Z "), and R 5 , R 6 , Y "', V, R, a, R 2 ' and B 'have the meaning given above, can be extended in the 5'3' direction.
  • the synthesis of building blocks XIII is carried out, for example, as in Seliger et al. (Nucleosides, Nucleotides 10 (1991), 469)
  • a (3'5'S) loop can be made up of multiple, preferably 4 to Introduce 5-fold successive coupling with the block of formula XIVb. After that the synthesis is continued in the 3'5 'direction as previously described.
  • nucleic acids preferably DNA
  • 5'5 'spacers of greater length.
  • a Triethylene glycol diphosphate on a chain built in 3'5 'direction or several times, preferably twice by coupling twice with the building block Formula XIVa can be installed at the desired location.
  • the Polarity is then synthesized using nucleotide building blocks of formula XIII 5'3 'direction continued.
  • a 3'3 'spacer is inserted at the intended location and then the Synthesis of the chain in the 3'5 'direction by condensation with Nucleoside 3'-phosphoramidites continued. It is a 3'3 'spacer
  • the introduction of a propane-1,3-diol diphosphate group can be introduced by coupling with the block of formula XIVd.
  • amino protective groups of the bases B 'and the nature of the linker arm A depend in individual cases on the nature of the substituent Z, since these must be easily cleavable after complete synthesis.
  • Z OiC 3 H 7
  • PAC labile phenoxyacetyl
  • conjugates have additional functional groups which must be appropriately protected before incorporation into the monomeric building blocks of the formulas V and XIII.
  • the carboxyl group of fluorescein must be protected as an alkyl ester.
  • the amide group can be present as an N-Fmoc (fluorenylmethoxycarbonyl) protected compound.
  • Hydroxy groups can be protected from side reactions by acylation or silylation (t-butyldimethylsilyl).
  • Amino groups can also be protected with trifluoroacetyl.
  • the conjugates may be so unstable that they would already be destroyed under the conditions of deprotection in oligonucleotide synthesis.
  • the synthesized oligonucleotide derivatives are characterized by Electro-spray ionization mass spectrometry (Stults and Masters, Rapid Commun. Measure Spectr. 5 (1991) 350).
  • oligonucleotide analogs of the formula IB according to the invention were based on tested their stability in serum and against known exonucleases.
  • oligonucleotide analogs of formula IB are also stable to Snake venom phosphodiesterase. Unmodified oligonucleotides are made by Snake venom phosphodiesterase from the 3 'end and spleen phosphodiesterase degraded exonucleolytically from the 5 'end.
  • the oligonucleotide analogs of formula IB form Watson-Crick base pairing with complementary single-stranded Nucleotide sequences of stable double-stranded hybrids or by Watson-Crick and Hoogsteen base pairing stable triplex structures while using double-stranded nucleic acids via Hoogsteen base pairing tripelhelicale Form structures, the spacers (5'5'S) and (3'3'S) changing strands enable.
  • Oligonucleotide analogs Formula IB can therefore be used for therapy or prophylaxis of virals Infections or cancer.
  • sequence IA-2 (example 4b)
  • sequence IA-2 (example 4b)
  • An oligonucleotide of formula IA-4 (example 4e) of sequence IV modified with psoralen at both 3 'ends recognizes the origin of replication (ori L ) of the HSV-1 genome and inhibits its replication by triplex formation.
  • the antiviral effectiveness of the psoralen conjugates can be significantly increased by irradiation with UV light.
  • the HSV-1 genome with its 160,000 bases naturally offers countless other target sequences of different efficiency to inhibit virus replication.
  • the therapeutic principle can also be applied to any other viruses, bacteria or other pathogens. The only requirement for a transfer to other disease triggers is that one knows the genes of these pathogens that are essential for the life cycle.
  • Sequence V is directed, for example, against the APP770 gene promoter, which is responsible for the expression of the precursor protein of the plaque-forming amyloid proteins in Alzheimer's disease.
  • Sequence VI simulates the SP1 binding region of the adenovirus E1b as a sense oligonucleotide and inhibits the transcription of E1b as a 3'-modified oligonucleotide analog with a 3'5 'spacer of the formula IB-2.
  • oncogenes are abl, new, myc, myb, ras, fos, mos, erbB, ets, jun, p53, src and rel.
  • Probes for nucleic acids in particular for DNA from oligonucleotide analogs Formula IA and B offer compared to the known oligonucleotide derivatives with a 3'-hydroxy group on the one hand the advantage of increased nuclease stability, on the other hand, they also allow the inclusion of the same or different ones Marker molecules at both ends of the oligonucleotide.
  • the advantage is that selectively different marker groups within an oligonucleotide Get stimulated (double marking).
  • the bifunctional derivatization can also used to place a marker on one end and the other end to introduce an additional function (e.g. affinity label).
  • To this Purpose can, for example, incorporate biotin at a 3 'end of the oligonucleotide that recognizes avidin or streptavidin, while at the other 3'-end an alkylamino linker attached an acridinium ester chemiluminescent marker can be.
  • the penetration behavior of the invention Oligonucleotide analogs cheaper than the unmodified oligonucleotides, especially when lipophilic residues have been introduced.
  • oligonucleotide analogs for example, also as aids in biotechnology and Molecular biology can be used.
  • the invention further relates to pharmaceutical preparations that are effective Amount of one or more compounds of formula IB or their physiologically acceptable salts, optionally together with physiologically compatible auxiliaries and / or carriers and / or together with others known active ingredients, as well as a method for producing them Preparations, characterized in that the active ingredient, together with the carrier and possibly other auxiliaries, additives or active ingredients in a suitable Dosage form brings.
  • the application is preferably intravenous, topical or intranasally.
  • 10 nmol of the oligonucleotide are to be examined in 450 ⁇ l 20% fetal calf serum in RPMI medium and 50 ml of double-distilled water and incubated at 37 ° C. Then immediately and after 1, 2, 4, 7 and 24 hours, 10 ⁇ l samples for gel electrophoresis or 20 ⁇ l samples for HPLC are taken, 5 ⁇ l or 10 ⁇ l formamide are added to terminate the reaction, and 5 Heated to 95 ° C for minutes. For gel electrophoresis, the samples are applied to a 15% polyacrylamide gel (2% BIS) and this is developed at about 3000 volt hours. The bands are made visible by the silver coloring.
  • 2% BIS polyacrylamide gel
  • the samples are sprayed onto a Gen-Pak Fax HPLC column (from Waters / Millipore) and chromatographed at 1 ml / min with 5 to 50% buffer A in B (buffer A: 10 mM sodium dihydrogen phosphate, 0.1 M NaCl in acetonitrile / water 1: 4 (v: v) pH 6.8; buffer B: as A, but 1.5 M NaCl).
  • the antiviral activity of the compounds according to the invention is in vitro Try tested.
  • the compounds according to the invention are described in different dilutions to cell cultures of HeLa and Vero cells in Given microtiter plates. After 3 hours, the cultures with different Human pathogenic viruses (e.g. herpes viruses HSV-1, HSV-2, orthomyxoviruses Influenza A2, Picornaviruses Rhinovirus 2) infected. 48 to 72 h after infection the success of therapy based on the cytopathogenic effect microscopic and after Neutral red image (color test after Finter) determined photometrically (Finter, N.B., In "Interferones", N.B. Finter et al., North Holland Publishing Co., Amsterdam, 1966). The minimum concentration at which about half of the infected cells do not show cytopathogenic effect is called minimal inhibitory concentration (MIC) considered.
  • MIC minimal inhibitory concentration

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EP01116471A 1992-01-22 1993-01-21 Analogues des oligonucléotides dérivées en position 3', avec des groupes non-nucléotidiques, leur préparation et leur utilisation Expired - Lifetime EP1142902B1 (fr)

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DE4201663 1992-01-22
DE4201663 1992-01-22
EP93100893A EP0552767B1 (fr) 1992-01-22 1993-01-21 Analogues des oligonucléotides dérivées en position 3', avec des groupes non-nucléotidics, leur préparation et leur utilisation

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US4739044A (en) * 1985-06-13 1988-04-19 Amgen Method for derivitization of polynucleotides
WO1988003149A1 (fr) * 1986-10-30 1988-05-05 Daicel Chemical Industries, Ltd. Procede de synthese d'oligonucleotides et composes servant a former des groupes protecteurs a poids moleculaire eleve
US4816571A (en) * 1987-06-04 1989-03-28 Applied Biosystems, Inc. Chemical capping by phosphitylation during oligonucleotide synthesis
US5216141A (en) * 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5399676A (en) * 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
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FI115214B (fi) * 1992-01-22 2005-03-31 Hoechst Ag Menetelmä oligonukleotidianalogien valmistamiseksi ja niiden käyttö

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HUT63172A (en) 1993-07-28
NO930200D0 (no) 1993-01-21
JP3676388B2 (ja) 2005-07-27
ZA93423B (en) 1993-09-09
FI930221A0 (fi) 1993-01-20
AU657191B2 (en) 1995-03-02
KR100273824B1 (ko) 2000-12-15
EP1142902B1 (fr) 2005-10-26
EP0552767B1 (fr) 2002-05-22
NO930200L (no) 1993-07-23
JPH05310778A (ja) 1993-11-22
DK0552767T3 (da) 2002-08-19
CA2087817A1 (fr) 1993-07-23
EP0552767A2 (fr) 1993-07-28
ATE217881T1 (de) 2002-06-15
ES2177533T3 (es) 2002-12-16
ATE307821T1 (de) 2005-11-15
DK1142902T3 (da) 2006-03-06
US5646261A (en) 1997-07-08
AU3191193A (en) 1993-07-29
PL172245B1 (pl) 1997-08-29
IL104460A (en) 2003-03-12
DE59310287D1 (de) 2002-06-27
ES2250268T3 (es) 2006-04-16
CA2087817C (fr) 2010-03-16
FI930221L (fi) 1993-07-23
FI115215B (fi) 2005-03-31
NZ245721A (en) 1995-09-26
EP0552767A3 (en) 1994-09-07
NO308609B1 (no) 2000-10-02
IL104460A0 (en) 1993-05-13
PT552767E (pt) 2002-10-31
KR930016438A (ko) 1993-08-26
DE59310380D1 (de) 2005-12-01
PL297516A1 (en) 1993-09-06
EP1142902A3 (fr) 2001-10-17
HU9300161D0 (en) 1993-04-28

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